Presence of integrons and their correlation with multidrug resistance in Salmonella enterica serovar Typhimurium: Exploratory systematic review / Presencia de integrones y su correlación con la multirresistencia en Salmonella enterica serovar Typhimurium: revisión sistemática exploratoria.

In Salmonella enterica serovar Typhimurium (Typhimurium), multidrug resistance is associated with integrons carrying resistance genes dispersed by mobile genetic elements. This exploratory systematic review sought to identify integron types and their resistance genes in multidrug resistance Typhimurium isolates. We used Medline, PubMed, SciELO, ScienceDirect, Redalyc, and Google Scholar as motor searchers for articles in Spanish or English published between 2012 and 2020, including the keywords "integrons", "antibiotic resistance", and "Salmonella Typhimurium". We included 38 articles reporting multidrug resistance up to five antibiotic families. Class 1 integrons with aadA2 and blaPSE-1 gene cassettes were predominant, some probably related to the Salmonella genomic island 1. We did not find studies detailing class 1 and 2 integrons in the same isolate, nor class 3 integrons reported. The presence of integrons largely explains the resistance profiles found in isolates from different sources in 15 countries.

La multirresistencia a los antibióticos en Salmonella enterica serovar Typhimurium (Typhimurium) se asocia con integrones que portan genes de resistencia y que son dispersados por elementos genéticos móviles. En esta revisión sistemática exploratoria, se buscó identificar los tipos de integrones y sus genes de resistencia en aislamientos de Typhimurium multirresistentes a antibióticos. Se realizó una búsqueda de artículos en Medline, PubMed, SciELO, ScienceDirect, Redalyc y Google Académico, publicados entre el 2012 y el 2020, en español o inglés, con las palabras claves: "integrons", "antibiotic resistance" y "Salmonella Typhimurium". En el análisis se incluyeron 38 artículos que reportaron multirresistencia a cinco familias de antibióticos. Los integrones de clase 1 con casetes de genes aadA2 y blaPSE-1 fueron los predominantes, algunos probablemente relacionados con la isla genómica de Salmonella 1. No se encontraron integrones de clase 1 y 2 en un mismo aislamiento, ni se reportaron integrones de clase 3. La presencia de integrones explica en gran medida los perfiles de resistencia encontrados en aislamientos de diferentes fuentes de 15 países.

Combined use of digestate and inorganic fertilizer alleviates the burden of class 1 integrons in perennial ryegrass rhizosphere without compromising aerial biomass production.

Antimicrobial resistance (AMR) is one of the main global health challenges. Anaerobic digestion (AD) can significantly reduce the burden of antibiotic resistance genes (ARGs) in animal manures. However, the reduction is often incomplete. The agronomic use of digestates requires assessments of their effects on soil ARGs. The objective of this study was to assess the effect of digestate on the abundance of ARGs and mobile genetic elements (MGEs) in the rhizosphere of ryegrass (Lolium perenne L.) and to determine whether half-dose replacement of digestate with urea (combined fertilizer) can be implemented as a safer approach while maintaining a similar biomass production. A greenhouse assay was conducted during 190 days under a completely randomized design with two experimental factors: fertilizer type (unfertilized control and fertilized treatments with equal N dose: digestate, urea and combined fertilizer) and sampling date (16 and 148 days after the last application). The results indicated that the digestate significantly increased the abundance of clinical class 1 integrons (intI1 gene) relative to the unfertilized control at both sampling dates (P < 0.05), while the combined fertilizer only increased them at the first sampling. Sixteen days after completing the fertilization scheme only the combined fertilizer and urea significantly increased the biomass production relative to the control (P < 0.05). Additionally, by the end of the assay, the combined fertilizer showed significantly lower levels of the macrolide-resistance gene ermB than digestate and a cumulative biomass similar to urea or digestate. Overall, the combined fertilizer can alleviate the burden of integrons and ermB while simultaneously improving biomass production.

Prevalence and characterization of quinolone resistance and integrons in clinical Gram-negative isolates from Gaza strip, Palestine.

BACKGROUND: Gram-negative bacteria with quinolone resistance and extended-spectrum beta-lactamases (ESBLs) present significant treatment challenges. This study evaluated the prevalence and characteristics of quinolone resistance in Gram-negative strains, investigating the relationship between plasmid-mediated quinolone resistance (PMQR), ESBLs, and integrons. METHODS AND RESULTS: We collected 146 Gram-negative isolates from patients in three Palestinian hospitals. For quinolone resistance isolates, the presence and characterization of PMQR, ß-lactamase genes and integrons were studied by PCR and sequencing. Out of 146 clinical isolates, 64 (43.8%) were resistant to quinolones, with 62 (97%) being multidrug-resistant (MDR) and 33 (51.5%) ESBL-producers. PMQR-encoding genes were present in 45 (70.3%) isolates, including aac(6')-Ib-cr (26.6%), qnrA (18.8%), qnrS1 (20.8%), and qnrB (6.4%). BlaCTX-M genes were detected in 50% (32/64) of isolates, with blaCTX-M-15 being the most common. BlaTEM-1, blaSHV-1 and blaVIM genes were found in 13, 6, and 4 isolates, respectively. Class I integrons were found in 31/64 (48%) of isolates, with 14 containing gene cassettes conferring resistance to trimethoprim (dhfr17, dfrA12, dfrA1) and aminoglycosides resistance genes (aadA1, aadA2, aadA5, and aadA6). CONCLUSIONS: This study found a high rate of quinolone resistance, ESBL and integrons in clinical Gram-negative isolates from our hospitals. Urgent measures are crucial, including implementing an antimicrobial resistance surveillance system, to control and continuously monitor the development of antimicrobial resistance.

Genetic features of BEL-1-producing and KPC-2-producing E. coli from hospital wastewater: human source or sewages adaptation.

Hospital sewage is an ecosystem that facilitates the transfer of antibiotic and heavy metal resistance genes and the interaction of human and environmental bacteria. In this environment, we have detected the presence of 7 KPC-2 and BEL-1 co-producing E. coli isolates of two different clones over a 10-month period in the same hospital. All isolates carried blaKPC-2 and the operon mer on the same IncP plasmid of similar size and an IncN plasmid of different size each clone carrying blaBEL-1. Both IncN-blaBEL-1 plasmids shared a 77 kb region containing blaBEL-1 alongside with fosE, bla OXA-10 and aac(6')-1b genes in a class 3 integron within a Tn3 transposon. The major IncN plasmid contained in addition a region homolog to P1-like bacteriophage RCS47, including the lytic RepL and lysogenic proteins, but other phage regions were incomplete. The characters such as the temporal persistence in sewage, the absence of colonized patients in the hospital or in the region, the presence of a p1 phage-plasmid fusion and the infrequent class 3 integron as genetic platform would indicate that BEL-1-producing isolates could have been generated in situ by adaptation to human sewage. Part of the microbiota in these discharges could be explained by the interactions of sewage ecosystems and not derive directly from the hospital.

In Silico Detection of Integrons and Their Relationship with Resistance Phenotype of Salmonella Isolates from a Brazilian Pork Production Chain.

The pork production chain is an important reservoir of antimicrobial resistant bacteria. This study identified and characterized integrons in Salmonella isolates from a Brazilian pork production chain and associate them with their antibiotic resistance pattern. A total of 41 whole-genome sequencing data of nontyphoidal Salmonella were analyzed using PlasmidSPAdes and IntegronFinder software. Nine isolates (21.9%) had some integrons identified (complete and/or incomplete). Six complete class 1 integrons were found, with streptomycin resistance genes (aadA1, aadA2) alone or downstream of a trimethoprim resistance gene (dfrA1, dfrA12), and some also containing resistance genes for sulfonamides (sul1, sul3) and chloramphenicol (cmlA1). Class 2 integron was detected in only one isolate, containing dfrA1-sat2-aadA1 gene cassettes. Five isolates harbored CALINs-clusters attC but lacking integrases-with antimicrobial resistance genes typically found in integron structures. In all, integrons were observed among four serotypes: Derby, Bredeney, Panama, and monophasic var. Typhimurium I 4,[5],12:i:-. The association of integrons with antibiotic resistance phenotype showed that these elements were predominantly identified in multidrug resistance isolates, and six of the seven gentamicin-resistant isolates had integrons. So, surveillance of integrons in Salmonella should be performed to identify the potential for the spread of antimicrobial resistance genes among bacteria.

Molecular Prevalence and Geographical Variations of Carbapenem-Resistant Klebsiella pneumoniae ST15 Isolates in a Tertiary Hospital in Ningbo, China.

BACKGROUND In China, the most prevalent type of CRKP is ST11, but the high-risk clone ST15 has grown in popularity in recent years, posing a serious public health risk. Therefore, we investigated the molecular prevalence characteristics of ST15 CRKP detected in a tertiary hospital in Ningbo to understand the current potential regional risk of ST15 CRKP outbreak. MATERIAL AND METHODS We collected and evaluated 18 non-duplicated CRKP strains of ST15 type for antibiotic resistance. Their integrons, virulence genes, and resistance genes were identified using polymerase chain reaction (PCR), and their homology was determined using MALDI-TOF MS. RESULTS The predominant serotype of 18 ST15 CRKP strains was K5. ST15 CRKP exhibited the lowest antimicrobial resistance to Cefoperazone/sulbactam (11.1%), followed by trimethoprim/sulfamethoxazole (22.2%). Resistance gene testing revealed that 14 out of 18 ST15 CRKP strains (77.8%) carried Klebsiella pneumoniae carbapenemase 2 (KPC-2), whereas all ST15 CRKP integrons were of the intI1 type. Furthermore, virulence gene testing revealed that all 18 ST15 CRKP strains carried ybtS, kfu, irp-1, and fyuA genes, followed by the irp-2 gene (17 strains) and entB (16 strains). The homology analysis report showed that 2 clusters had closer affinity, which was mainly concentrated in classes C and D. CONCLUSIONS The ST15 CRKP antibiotic resistance rates demonstrate clear geographical differences in Ningbo. Additionally, some strains carried highly virulent genes, indicating a possible evolution towards carbapenem-resistant highly virulent strains. To reduce the spread of ST15 CRKP, we must rationalize the clinical use of antibiotics and strengthen resistance monitoring to control nosocomial infections.

Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics.

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

Characterization of biofilm formation and multi-drug resistance among Pseudomonas aeruginosa isolated from hospital wastewater in Dhaka, Bangladesh.

Hospital wastewater has been identified as a hotspot for the emergence and transmission of multidrug-resistant (MDR) pathogens that present a serious threat to public health. Therefore, we investigated the current status of antibiotic resistance as well as the phenotypic and genotypic basis of biofilm formation in Pseudomonas aeruginosa from hospital wastewater in Dhaka, Bangladesh. The disc diffusion method and the crystal violet assay were performed to characterize antimicrobial resistance and biofilm formation, respectively. Biofilm and integron-associated genes were amplified by the polymerase chain reaction. Isolates exhibited varying degrees of resistance to different antibiotics, in which >80% of isolates showed sensitivity to meropenem, amikacin, and gentamicin. The results indicated that 93.82% of isolates were MDR and 71 out of 76 MDR isolates showed biofilm formation activities. We observed the high prevalence of biofilm-related genes, in which algD+pelF+pslD+ (82.7%) was found to be the prevalent biofilm genotypic pattern. Sixteen isolates (19.75%) possessed class 1 integron (int1) genes. However, statistical analysis revealed no significant association between biofilm formation and multidrug resistance (χ2 = 0.35, P = 0.55). Taken together, hospital wastewater in Dhaka city may act as a reservoir for MDR and biofilm-forming P. aeruginosa, and therefore, the adequate treatment of wastewater is recommended to reduce the occurrence of outbreaks.

Analysis of the Class 1 Integrons, Carbapenemase Genes and Biofilm Formation Genes Occurrence in Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is a non-fermentative Gram-negative bacterium that can cause nosocomial infections in critically ill patients. Carbapenem-resistant A. baumannii (CRAB) has spread rapidly in clinical settings and has become a key concern. The main objective of this study was to identify the distribution of integrons and biofilm-formation-related virulence genes in CRAB isolates. A total of 269 A. baumannii isolates (219 isolates of CRAB and 50 isolates of carbapenem-sensitive A. baumannii (CSAB)) were collected. Carbapenemase genes (bla KPC, bla VIM, bla IMP, bla NDM, and bla OXA-23-like) and biofilm-formation-related virulence genes (abal, bfms, bap, and cusE) were screened with PCR. Class 1 integron was screened with PCR, and common promoters and gene cassette arrays were determined with restriction pattern analysis combined with primer walking sequencing. Whole-genome sequencing was conducted, and data were analyzed for a bla OXA-23-like-negative isolate. All 219 CRAB isolates were negative for bla KPC, bla VIM, bla IMP, and bla NDM, while bla OXA-23-like was detected in 218 isolates. The detection rates for abal, bfms, bap, and cusE in 219 CRAB were 93.15%, 63.93%, 88.13%, and 77.63%, respectively. Class 1 integron was detected in 75 CRAB (34.25%) and in 3 CSAB. The single gene cassette array aacA4-catB8-aadA1 with relatively strong PcH2 promoter was detected in class 1 integrons. The bla OXA-23-like-negative CRAB isolate was revealed to be a new sequence type (Oxford 3272, Pasteur 2520) carrying bla OXA-72, bla OXA-259, and bla ADC-26. In conclusion, bla OXA-23-like was the main reason for CRAB's resistance to carbapenems. A new (Oxford 3272, Pasteur 2520) CRAB sequence type carrying the bla OXA-72, bla OXA-259, and bla ADC-26 was reported.

Distribution and expression of the aac(6')-Im (aacA16) aminoglycoside resistance gene.

BACKGROUND: The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. OBJECTIVES: To identify sources for the aac(6')-Im fragment found in A. baumannii. METHODS: MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. RESULTS: Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different. CONCLUSIONS: The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.

Emergence of extensive drug resistance and high prevalence of multidrug resistance among clinical Proteus mirabilis isolates in Egypt.

BACKGROUND: Proteus mirabilis is an opportunistic pathogen that has been held responsible for numerous nosocomial and community-acquired infections which are difficult to be controlled because of its diverse antimicrobial resistance mechanisms. METHODS: Antimicrobial susceptibility patterns of P. mirabilis isolates collected from different clinical sources in Mansoura University Hospitals, Egypt was determined. Moreover, the underlying resistance mechanisms and genetic relatedness between isolates were investigated. RESULTS: Antimicrobial susceptibility testing indicated elevated levels of resistance to different classes of antimicrobials among the tested P. mirabilis clinical isolates (n = 66). ERIC-PCR showed great diversity among the tested isolates. Six isolates (9.1%) were XDR while all the remaining isolates were MDR. ESBLs and AmpCs were detected in 57.6% and 21.2% of the isolates, respectively, where blaTEM, blaSHV, blaCTX-M, blaCIT-M and blaAmpC were detected. Carbapenemases and MBLs were detected in 10.6 and 9.1% of the isolates, respectively, where blaOXA-48 and blaNDM-1 genes were detected. Quinolone resistant isolates (75.8%) harbored acc(6')-Ib-cr, qnrD, qnrA, and qnrS genes. Resistance to aminoglycosides, trimethoprim-sulfamethoxazole and chloramphenicol exceeded 80%. Fosfomycin was the most active drug against the tested isolates as only 22.7% were resistant. Class I or II integrons were detected in 86.4% of the isolates. Among class I integron positive isolates, four different gene cassette arrays (dfrA17- aadA5, aadB-aadA2, aadA2-lnuF, and dfrA14-arr-3-blaOXA-10-aadA15) and two gene cassettes (dfrA7 and aadA1) were detected. While class II integron positive isolates carried four different gene cassette arrays (dfrA1-sat1-aadA1, estXVr-sat2-aadA1, lnuF- dfrA1-aadA1, and dfrA1-sat2). CONCLUSION: P. Mirabilis ability to acquire resistance determinants via integrons may be held responsible for the elevated rates of antimicrobial resistance and emergence of XDR or even PDR strains limiting the available therapeutic options for management of infections caused by those strains.

Bacterial dynamics of the plastisphere microbiome exposed to sub-lethal antibiotic pollution.

BACKGROUND: Antibiotics and microplastics are two major aquatic pollutants that have been associated to antibiotic resistance selection in the environment and are considered a risk to human health. However, little is known about the interaction of these pollutants at environmental concentrations and the response of the microbial communities in the plastisphere to sub-lethal antibiotic pollution. Here, we describe the bacterial dynamics underlying this response in surface water bacteria at the community, resistome and mobilome level using a combination of methods (next-generation sequencing and qPCR), sequencing targets (16S rRNA gene, pre-clinical and clinical class 1 integron cassettes and metagenomes), technologies (short and long read sequencing), and assembly approaches (non-assembled reads, genome assembly, bacteriophage and plasmid assembly). RESULTS: Our results show a shift in the microbial community response to antibiotics in the plastisphere microbiome compared to surface water communities and describe the bacterial subpopulations that respond differently to antibiotic and microplastic pollution. The plastisphere showed an increased tolerance to antibiotics and selected different antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs). Several metagenome assembled genomes (MAGs) derived from the antibiotic-exposed plastisphere contained ARGs, virulence factors, and genes involved in plasmid conjugation. These include Comamonas, Chryseobacterium, the opportunistic pathogen Stenotrophomonas maltophilia, and other MAGs belonging to genera that have been associated to human infections, such as Achromobacter. The abundance of the integron-associated ciprofloxacin resistance gene aac(6')-Ib-cr increased under ciprofloxacin exposure in both freshwater microbial communities and in the plastisphere. Regarding the antibiotic mobilome, although no significant changes in ARG load in class 1 integrons and plasmids were observed in polluted samples, we identified three ARG-containing viral contigs that were integrated into MAGs as prophages. CONCLUSIONS: This study illustrates how the selective nature of the plastisphere influences bacterial response to antibiotics at sub-lethal selective pressure. The microbial changes identified here help define the selective role of the plastisphere and its impact on the maintenance of environmental antibiotic resistance in combination with other anthropogenic pollutants. This research highlights the need to evaluate the impact of aquatic pollutants in environmental microbial communities using complex scenarios with combined stresses. Video Abstract.

Screening Key Sites of Class 2 Integron Integrase that Impact Recombination Efficiency.

By capturing and expressing exogenous resistance gene cassettes through site-specific recombination, integrons play important roles in the horizontal transfer of antimicrobial resistant genes among bacteria. The characteristics of integron integrase make it to be a potential gene editing tool enzyme. In this study, a random mutation library using error-prone PCR was constructed, and amino acid residues mutants that impact on attI2 × attC or attC × attC recombination efficiency were screened and analyzed. Thirteen amino acid mutations were identified to be critical impacted on site-specific recombination of IntI2, including the predicted catalyzed site Y301. Nine of 13 mutated amino acid residues that have critically impacted on IntI2 activity were relative concentrated and near the predicted catalyzed site Y301 in the predicted three-dimensional structure indicated the importance of this area in maintain the activity of IntI2. No mutant with obviously increased recombination activity (more than four-fold as high as that of wild IntI2) was found in library screening, except P95S, R100K slightly increased (within two-fold) the excision activity of IntI2, and S243T slightly increased (within two-fold) both excision and integration activity of IntI2. These findings will provide clues for further specific modification of integron integrase to be a tool enzyme as well as establishing a new gene editing system and applied practically.

Evaluation of the presence of integrons, sul and smqnr genes and the prevalence of antibiotic resistance in Stenotrophomonas maltophilia clinical isolates.

OBJECTIVES: The objective of this investigation was to examine the mechanisms associated with antibiotic resistance in Stenotrophomonas maltophilia clinical isolates retrieved from hospitalized patients undergoing open heart surgery in a Heart Center located in Tehran, Iran. MATERIALS AND METHODS: This investigation encompassed a cross-sectional study of 60 S. maltophilia isolates, which were procured from diverse clinical specimens. Primary identification of the isolates was conducted through conventional microbiologic methods and subsequently verified by means of PCR primers. The E-test was utilized to establish the minimum inhibitory concentrations (MICs). PCR was then employed to ascertain the antibiotic resistance genes (sul1, sul2, Smqnr and intl1 - intl3). RESULTS: In this study, a total of sixty clinical isolates of S. maltophilia were collected, with the majority of them being obtained from Intensive Care Units (ICU) (n = 54; 90%). The disk diffusion method yielded results indicating that 55% of the isolates were sensitive to minocycline, whereas 30% were intermediate and 15% were found to be resistant. Additionally, the MIC results revealed that the resistant rates of the isolates towards ceftazidime, cotrimoxazole and levofloxacin were 46.7%, 1.7% and 5%, respectively. The PCR amplification of three classes of integrons genes indicated that fifteen (25%) of the isolates carried int1, while no detection for intl2 and intl3 was reported. Furthermore, the prevalence of antibiotic resistance genes (sul1, sul2, and Smqnr) was identified in 15 (25%), 6 (10%), and 28 (46.7%) isolates, respectively. CONCLUSION: The reported increasing rate of antibiotic resistance and mobile genetic elements that could extend the resistance genes to other strains in the hospital, finally it could be an alarming issue for healthcare settings that need special attention to this strain and the epidemiological study on this issue.

Transmission of antibiotic resistance through organic amendments in arable land: A 3-year field study with pigeonpea-wheat cropping system.

The worldwide emergence of antimicrobial resistance (AMR) poses a substantial risk to human health and environmental stability. In agriculture, organic amendments (derived from organic sources such as manure, and plant residues) are beneficial in restoring soil properties and providing essential nutrients to crops but raise concerns about harboring antibiotic resistance, which emphasizes the need for vigilant monitoring and strategic interventions in their application. The current study assessed the impact of farming practices (organic and conventional) in a three-year field experiment with pigeonpea-wheat cropping system, focusing on the transmission of AMR using culture-dependent and -independent approaches, and soil nutrient content. Markers for antibiotic resistance genes (ARGs) (aminoglycoside-aacA, ß-lactam-blaTEM, chloramphenicol-cmlA1, macrolide-ermB, sulfonamides-sul1, sul2, and tetracycline-tetO) and integrons (intl1 and intl2) were targeted using qPCR. Manure amendments, particularly FYM1, exhibited a higher abundance of copies of ARGs compared to the rhizospheric soil. Organic farming was associated with higher copies of intl2, sul1, blaTEM, and tetO genes, while conventional farming showed increased copies of sul2 and ermB genes in the rhizosphere. Significant positive correlations were observed among soil nutrient contents, ARGs, and MGEs. The notable prevalence of ARGs linked to manure amendments serves as a cautionary note, demanding responsible management practices.

Integron-associated genes are reliable indicators of antibiotic resistance in wastewater despite treatment- and seasonality-driven fluctuations.

The present study aims to characterize the bacterial community, resistome and integron abundance of a municipal wastewater treatment plant (WWTP) over the course of 12 months and evaluate the year-long performance of integron-related genes as potential indicators of antibiotic resistance mechanisms in influents and effluents. For that, total DNA was extracted and subjected to 16S rRNA-targeted metabarcoding, high-throughput (HT) qPCR (48 targets) and standard qPCR (5 targets). Targets included integrase genes, antibiotic resistance genes (ARGs) and putative pathogenic groups. A total of 16 physicochemical parameters determined in the wastewater samples were also considered. Results revealed that the WWTP treatment significantly impacted the bacterial community, as well as the content in ARGs and integrase genes. Indeed, there was a relative enrichment from influent to effluent of 13 pathogenic groups (e.g., Legionella and Mycobacterium) and genes conferring resistance to sulphonamides, aminoglycosides and disinfectants. Effluent samples (n = 25) also presented seasonal differences, with an increase of the total ARGs' concentration in summer, and differences between winter and summer on relative abundance of sulphonamide and disinfectant resistance mechanisms. From the eight putative integron-related genes selected, all were positively correlated with the total ARGs' content in wastewater and the relative abundance of resistance to most of the specific antibiotic classes. The genes intI1, blaGES and qacE∆1 were the most strongly correlated with the total concentration of ARGs. Genes blaGES and blaVIM, were better correlated to resistance to beta-lactams, aminoglycosides and tetracyclines. This study supports the use of integron-related genes as powerful indicators of antibiotic resistance in wastewater, being robust despite the variability caused by wastewater treatment and seasonality.

Belt and braces: Two escape ways to maintain the cassette reservoir of large chromosomal integrons.

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated with a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context. Through experimental evolution, we find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate a nice interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs.

[Molecular Analysis of Antibiotic Resistance in CarbapenemResistant Pseudomonas aeruginosa Isolates Isolated from Clinical Samples]. / Klinik Örneklerden Izole Edilen Karbapenem Dirençli Pseudomonas aeruginosa Izolatlarindaki Antibiyotik Direncinin Moleküler Analizi.

Pseudomonas aeruginosa is an opportunistic pathogen that causes increased morbidity and mortality in risky patient groups. Nowadays, carbapenem resistance has become a threat and resistance genes are spreading among species through mobile genetic elements. The dissemination of carbapenemases among P.aeruginosa is a serious public health concern due to its limited options for the treatment of bacterial infections. In this study, it was aimed to investigate the molecular epidemiology of 47 carbapenem resistant P.aeruginosa (CRPA) isolates derived from various clinical samples from the Central Laboratory Bacteriology Unit of Kocaeli University Research and Training Hospital between October 2021 and March 2023. The rates of resistance to the antibiotics, some carbapenemase and virulence genes, conjugative resistance plasmids, integron gene cassette contents and the clonal similarity of the isolates were investigated and then epidemiologically evaluated. In the study, identification of the bacterial isolates and their susceptibility to some antibiotics (imipenem, meropenem, aztreonam, amikacin, netilmicin, tobramycin, piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, ciprofloxacin and levofloxacin) were determined by the VITEK® 2 Compact automated system. Metallo-beta-lactamase (MBL) production of the isolates was demonstrated by the imipenem/meropenem-EDTA (IMP/MEM-EDTA) combined disc method. Conjugation experiments were performed by the broth mating method. Alkali lysis method was used in plasmid DNA isolations. Co-transferred antibiotic resistances in transconjugants were detected by disc diffusion method. Carbapenemase genes (blaIMP , blaVIM , blaNDM , blaKPC and blaOXA-48 ), integron gene cassettes (class 1 and class 2) and virulence genes (lasR and rhlR) were screened by specific polymerase chain reactions (PCRs). Clonal relationships of the CRPA isolates were investigated by evaluating the DNA f ingerprintings obtained from the ERIC (enterobacterial repetitive intergenic consensus)-PCR assay. The highest resistance rate of the isolates were to levofloxacin, while the lowest resistance rates were observed against tobramycin, gentamicin and amikacin. MBL production was detected in 25 (53.2%) isolates. In conjugation experiments, 12 (25.5%) isolates were detected to harbour conjugative resistance plasmids. In 90% of the CRPA isolates, lasR and rhlR biofilm genes (encoding for the transcriptional activator protein) were detected by PCR. The blaVIM gene was detected in six (12.8%) isolates. The blaNDM gene was detected in five (10.6%) isolates and the blaOXA-48 gene was detected in three (6.4%) isolates. The blaKPC and blaIMP genes were not detected in CRPA isolates. It was determined that two (16.6%) of the isolates that carried the blaVIM gene, one (8.3%) carried the blaNDM gene and one (8.3%) carried the blaOXA-48 gene contained conjugative plasmids.In integron-specific PCRs, intI1 gene was positive in 39 (82.9%) isolates, while class 1 integron gene cassettes were detected in 24 isolates (51%). IntI1 positive six isolates were found to harbour class 1 integron gene cassettes-bearing conjugative plasmids. Class 2 integrons were not found in the CRPA isolates. Dendrogram analysis of ERIC-PCR patterns showed that there was no clonal similarity between the CRPA isolates and the isolates did not spread by cross-contamination. As a result, it has been observed that most of the CRPA isolates which have the potential to form biofilms, are highly resistant to other antibiotic groups other than carbapenems and can co-transfer some resistances (ceftazidime, cefepime, ciprofloxacin, levofloxacin, piperacillin-tazobactam) with conjugative resistance plasmids. It is thought that it would be useful to follow molecular epidemiology in the resistance gene reservoirs of these strains which have the potential to cause epidemics in the clinical arena.

Molecular Epidemiology of Carbapenem-Resistant Klebsiella aerogenes in Japan.

Information regarding Klebsiella aerogenes haboring carbapenemase in Japan is limited. A comprehensive nationwide survey was conducted from September 2014 to December 2022, and 67 non-duplicate strains of carbapenem-resistant K. aerogenes were isolated from 57 healthcare facilities in Japan. Through genetic testing and whole-genome sequencing, six strains were found to possess carbapenemases, including imipenemase (IMP)-1, IMP-6, New Delhi metallo-ß-lactamase (NDM)-1, and NDM-5. The strain harboring blaNDM-5 was the novel strain ST709, which belongs to the clonal complex of the predominant ST4 in China. The novel integron containing blaIMP-1 featured the oxacillinase-101 gene, which is a previously unreported structure, with an IncN4 plasmid type. However, integrons found in the strains possessing blaIMP-6, which were the most commonly identified, matched those reported domestically in Klebsiella pneumoniae, suggesting the prevalence of identical integrons. Transposons containing blaNDM are similar or identical to the transposon structure of K. aerogenes harboring blaNDM-5 previously reported in Japan, suggesting that the same type of transposon could have been transmitted to K. aerogenes in Japan. This investigation analyzed mobile genetic elements, such as integrons and transposons, to understand the spread of carbapenemases, highlighting the growing challenge of carbapenem-resistant Enterobacterales in Japan and underscoring the critical need for ongoing surveillance to control these pathogens.

Variations in antimicrobial resistance genes present in the rectal faeces of seals in Scottish and Liverpool Bay coastal waters.

Antibiotic resistance genes originating from human activity are considered important environmental pollutants. Wildlife species can act as sentinels for coastal environmental contamination and in this study we used qPCR array technology to investigate the variety and abundance of antimicrobial resistance genes (ARGs), mobile genetic elements (MGEs) and integrons circulating within seal populations both near to and far from large human populations located around the Scottish and northwest English coast. Rectal swabs were taken from 50 live grey seals and nine live harbour seals. Nucleic acids were stabilised upon collection, enabling extraction of sufficient quality and quantity DNA for downstream analysis. 78 ARG targets, including genes of clinical significance, four MGE targets and three integron targets were used to monitor genes within 22 sample pools. 30 ARGs were detected, as well as the integrons intl1 and intl2 and tnpA transposase. Four ß-lactam, nine tetracycline, two phenicol, one trimethoprim, three aminoglycoside and ten multidrug resistance genes were detected as well as mcr-1 which confers resistance to colistin, an important drug of last resort. No sulphonamide, vancomycin, macrolide, lincosamide or streptogramin B (MLSB) resistance genes were detected. Resistance genes were detected in all sites but the highest number of ARGs (n = 29) was detected in samples derived from grey seals on the Isle of May, Scotland during the breeding season, and these genes also had the highest average abundance in relation to the 16S rRNA gene. This pilot study demonstrates the effectiveness of a culture-independent workflow for global analysis of ARGs within the microbiota of live, free-ranging, wild animals from habitats close to and remote from human habitation, and highlights seals as a valuable indicator species for monitoring the presence, abundance and land-sea transference of resistance genes within and between ecosystems.